The present invention relates to systems for the electrophoretic separation of materials present in a sample wherein the materials have a range of molecular weights. The sample is applied to a gel or similar medium, e.g., on a tray in a vessel of buffer solution, and is subjected to an electric field for a time effective to separate components thereof. In particular the invention provides a stable multi-tray system for simultaneously processing multiple samples without the use of a mechanical buffer circulation pump.
Electrophoresis is the migration of electrically charged particles in solution or suspension under the influence of an applied electric field. Each charged particle moves toward the electrode of opposite electrical polarity to its charge. For a given set of solution conditions, the velocity with which a particle moves divided by the magnitude of the electric field is a characteristic number called the electrophoretic mobility. The electrophoretic mobility is directly proportional to the magnitude of the charge on the particle, and is inversely proportional to the size of the particle. This property has been used to determine protein molecular weights; to distinguish among molecules by virtue of their individual net electrical charge; to detect amino acid changes from charged to uncharged residues or vice versa; and to separate different molecular weight species quantitatively as well as qualitatively. In particular, electrophoresis is widely used to separate, characterize and identify large molecules such as proteins and linear molecules such as DNA and RNA, and strands and fragments thereof. More complete and detailed information regarding the basic types of electrophoresis and the many applications for which each type of electrophoresis is utilized may be found in Freifelder, D., Physical Biochemistry, Applications to Biochemistry and Molecular Biology, 2nd edition, W. H. Freeman and Company, New York, 1982, pages 276-322.
The resolving power of electrophoresis can be greatly improved by the use of gel supporting media. Gel electrophoresis methods and electrophoretic apparatus which utilize gels such as starch, polyacrylamide, agarose, and agarose-acrylamide as supporting media are well established. The variety of different applications and the value of gel electrophoresis as a superior analytical and/or preparative tool is demonstrated by the many innovations in apparatus for electrophoresis. These are exemplified by U.S. Pat. Nos. 3,047,489; 4,234,400; 4,151,065; 3,980,546; 3,980,540; 3,932,265; and 3,553,097. Generally, agarose and polyacrylamide are the main types of gels used for electrophoretic analysis of nucleic acid. Typically, short to intermediate size fragments (below one thousand base pairs) are separated in polyacrylamide, while intermediate to high molecular weight fragments are separated in agarose gels. Often, the polyacrylamide gel is arranged in a vertical format or column, while the agarose gels are arranged on horizontal trays in slabs or beds. Typically, to obtain a good separation of DNA and other large molecular weight compositions (proteins and nucleic acids), electrophoresis must be performed on an extended time basis. One of the constant problems of electrophoretic analysis is the breakdown of the buffer used to control the pH of the gel medium due to the formation of acid (H+) at the anode and the formation of base (OHxe2x88x92) at the cathode over time. This problem is usually resolved by using a mechanical pump for circulating the buffer fluid between the anode containing buffer chamber and the cathode containing buffer chamber.
One electrophoresis device using a buffer circulation system is Applicant""s previously patented gel electrophoresis apparatus shown in U.S. Pat. No. 4,702,814. That device has no mechanical circulation structure. It makes use of a pair of platinum electrodes, that are mounted in two buffer reservoirs provided at opposed ends of the device, and a tray supporting a gel bed positioned in a channel of fluid extending between the ends so that an electric field is established along the length of the channel. A hood is positioned below the surface of the buffer solution and covers the length of one of the end electrodes to capture evolved gas bubbles, and a fluid transfer passage extends from the hood at one reservoir upward and laterally to the reservoir at the other end of the apparatus. Bubbles of gas evolved from the underlying electrode enter the passage and drive buffer fluid from one reservoir to the other, creating an equalizing fluid transfer that prevents breakdown of the pH in both buffer reservoirs. The rate of transfer increases with the amount of evolved gas, and operates to transport the higher pH buffer from the cathode end and mix it with the lower pH buffer at the anode end, thus maintaining the buffer pH essentially constant throughout the whole system. The gas-driven circulation also promotes uniformity of temperature, so that the field separation characteristics remain constant and the band spacing is not subject to non-linear distortions at the central portion of the gel tray.
Other patents and marketed systems employ electromechanical pumps to effect buffer circulation and maintain substantial uniformity throughout the gel tray or trays. However, these systems are costly; the cost of a pump alone may be many hundreds of dollars. This is largely because the pump components must comply with fairly stringent standards of construction: the pump must be explosion-proof, electrically isolated, and emission-free for use in biological or laboratory situations.
While these devices have proven to be effective, recombinant DNA technologies, and the availability of polymerase chain reaction technology to amplify and produce effective sample sizes of arbitrary isolates, have expanded greatly the number of electrophoretic analyses performed, and have broadened the scope of such analyses to encompass broad applications in laboratory, industrial and clinical settings. As a result, greater throughput is needed in electrophoretic devices so that greater numbers of analyses can be run without requiring larger capital investments or taking up further space on testing benches.
Various techniques have been employed to refine the resolution or speed of the electrophoretic process for certain specific classes of molecules or conditions. For example, pulse field electrophoresis may be applied to better resolve very large DNA fragments, and extended gel lengths may be used to achieve finer resolution. Other techniques may involve the use of relatively low viscosity gels immobilized in capillary tubes for effecting the separation; the use of high field gradient, short path configurations; or the use of gradient gel concentration arrangements. In addition to having different column, capillary or bed formats, each gel material may present different effective porosity and characteristic ion mobilities. By way of example, the concentration of agarose used in the gel for DNA separations may vary from about 2% for separation of nucleic acid fragment under several thousand nucleotides in length, down to about 0.3% for separation of larger fragments having a size in the range of five thousand to sixty-thousand nucleotides. Similarly for polyacrylamide gels, the concentrations may range between about 3% for one-hundred to two-thousand nucleotide chains, up to about 20% for separating smaller molecules of lengths between about five and about one hundred nucleotides.
In electrophoretic separations, run lengths in vertical or horizontal format may range from about ten to about one-hundred centimeters, depending on the fragment size and the required degree of resolution. The voltage gradients may generally be about one to five volts per centimeter. Typically, a separation may take from one to about twenty hours depending upon resolution requirements and other parameters.
Various other techniques have been proposed to enhance speed or efficiency, or to more effectively integrate the separation process into a desired assay such as a DNA analysis. One such construction applies high electric fields, on the order of about five to about one-hundred volts per millimeter along the length of the gel axis in a relatively thin, e.g., (0.1 to 1.5 millimeter thick) gel bed, so as to effect fast separation over a length of under about two centimeters. Other thin bed techniques are used with greater length slabs, e.g., as in the Southern blot technique, in conjunction with other layers effective to transfer the bands of material, once separated, onto a blotter with high yield from the gel medium.
However, while the mechanism of gel electrophoresis allows a great many specialized variations, some of which can increase the speed of analysis in certain circumstances, the preponderance of separations in a working laboratory require a high-throughput separation apparatus that is both robust and inexpensive.
The present invention provides a device and method for carrying out electrophoretic separation of sample mixtures in a plurality of gels at the same time. The device of the invention provides increased throughput for separation analyses while taking up minimal space on a laboratory testing bench and requiring minimal capital investment as the device of the invention has a footprint no larger than existing single tray electrophoresis devices and requires no mechanical circulation pump.
The device of the invention includes a vessel containing buffer solution and having first and second end reservoirs and an intermediate channel fluidically interconnecting the two end reservoirs and configured to hold a stack of gel trays covereded by the buffer solution. A pair of electrodes, e.g., platinum wire electrodes, are mounted in the respective reservoirs at opposed ends of the vessel so that an oriented electric field is established along the length of the channel and in the trays between the reservoirs. Further, a gas driven recirculation assembly pumps the buffer from one end reservoir into buffer at the other end reservoir at a level below the level of the trays.
Preferably, the recirculation assembly is implemented with a hood that is positioned below the surface of the buffer solution and covering at least one of the electrodes to capture evolved gas bubbles, and a transfer passage that extends from a position near the top of the hood to the other end reservoir. Bubbles of gas evolved from the underlying electrode enter the passage and drive buffer fluid along the transfer passage from one end to the opposite end of the device, creating an equalizing fluid transfer that effectively neutralizes the fluid passing through the channel and over the gel trays. This prevents excessive buildup of acid or base in both buffer reservoirs and prevents breakdown of the buffer solution or the sample material. The rate of recirculation increases with the amount of evolved gas, and operates to transport the higher pH buffer from the cathode end and mix it with the lower pH buffer at the anode end, thus maintaining the buffer pH essentially constant. The gas-driven circulation is arranged with a return flow over the stacked gels, uniformizing temperature, so that electrophoretic field separation characteristics remain constant and the separation band spacing is not subject to non-linear distortions at the central portion of the gel tray due to temperature and/or pH gradients.
In a preferred embodiment, the gel trays are stackable, modular trays that each have one or more male registration elements on one or more sides, together with corresponding female registration elements, positioned in registry so that trays stack in alignment on one another without slipping or shifting. The stacked trays increase capacity of the device over the prior art without increasing footprint of the separation device, effectively multiplying separation throughput with minimal capital cost. The registration elements may be shaped such that the stacked trays have continuous confining sidewalls that effectively channel flow of buffer across the surface of each tray. The trays can also be dimensioned such that wall height provides a buffer spacing between layers that is comparable or somewhat less than the gel thickness. For example, a tray may have side walls about 1.5-2.0 centimeters high to support a gel layer one-half to about one centimeter thick leaving a 0.5 centimeter buffer space or recirculation return passage below the next higher tray. The trays may be formed of a suitable polymer, which may for example have a thickness of about 0.5 to 1.0 centimeters, so that at least one third and up to as much as one half of the volume of channel is occupied by gel layers and at least about one half of the volume of channel is occupied by the gel layers and gel supporting portions of the trays so that multiple trays may be stacked with sufficient recirculation flow to maintain generally uniform temperatures in the gel layers with only a nominal increase in buffer volume.